Microfluidic devices and systems provide improved methods of performing chemical, biochemical and biological analysis and synthesis: Microfluidic devices and systems allow for the performance of multi-step, multi-species chemical operations in chip-based micro chemical analysis systems. Chip-based microfluidic systems generally comprise conventional ‘microfluidic’ elements, particularly capable of handling and analyzing chemical and biological specimens. Typically, the term microfluidic in the art refers to systems or devices having a network of processing nodes, chambers and reservoirs connected by channels, in which the channels have typical cross-sectional dimensions in the range between about 1.0 μm and about 500 μm. In the art, channels having these cross-sectional dimensions are referred to as ‘microchannels’.
In the chemical, biomedical, bioscience and pharmaceutical industries, it has become increasingly desirable to perform large numbers of chemical operations, such as reactions, separations and subsequent detection steps, in a highly parallel fashion. The high throughput synthesis, screening and analysis of (bio)chemical compounds, enables the economic discovery of new drugs and drug candidates, and the implementation of sophisticated medical diagnostic equipment. Of key importance for the improvement of the chemical operations required in these applications are an increased speed, enhanced reproducibility, decreased consumption of expensive samples and reagents, and the reduction of waste materials.
In the fields of biotechnology, and especially cytology and drug screening, there is a need for high throughput filtration of particles. Examples of particles that require filtration are various types of cells, such as blood platelets, white blood cells, tumorous cells, embryonic cells and the like. These particles are especially of interest in the field of cytology. Other particles are (macro) molecular species such as proteins, enzymes and poly-nucleotides. This family of particles is of particular interest in the field of drug screening during the development of new drugs.
Proteomics is one of the main areas of modern biology, and has recently gained significant importance in biology, primarily because proteins are involved in virtually every cellular function, control every regulatory mechanism and are modified in disease (as a cause or effect). The proteome consists of all proteins present in a complex sample at a given time, including proteins translated directly from genetic material and a variety of modified proteins. These modified proteins can arise from alternative splicing of transcripts, or by extensive post-translation modifications (such as glycosylation, amidation, ubiquitination, phosphorylation, methylation) or by a combination of the two resulting in modifications that alter the function and/or structure of the protein. An aspect of proteomics often called “protein expression profiling” involves the qualitative and quantitative study of protein expression in samples that differ by some variable. Protein expression profiling has been used to find markers for disease states, to understand signaling networks in cells, and to look for new targets for drug design. The combination of high resolution separations and mass spectrometry (MS) analysis of peptide fragments have resulted in great ability to take a sample (from tissue, cells, serum, or subcellular fraction) and identify many, or all, of the proteins present, and gain an understanding of their state and abundance.
Available state-of-the-art analytical methodologies for protein expression profiling require extensive sample preparation and have complex sample handling requirements. Current technologies are also limited in their ability to recognize alterations in functional and structural properties of proteins (e.g. various states of post-translational modification). Therefore, there is an urgent need to develop a new class of analytical methodologies that can provide accurate, sensitive and more detailed information than available in current approaches.
Two common approaches exist to make use of MS identification of peptides in protein profiling. In one type of protein profiling (known as “digest-before-separate”), one takes the protein sample and digests the mixture down to peptide fragments, followed by liquid chromatography (LC) separation of the fragments and MS—MS analysis of the fragment sequence. In the other type (known as “separate-before-digest”), a protein mixture is separated by high resolution 2D Electrophoresis (2DE) (currently immobilized pH gradient (IPG) isoelectric focusing followed by SDS-PAGE) and then protein spots on the final 2D gel are excised, digested, and deposited on a surface for MALDI-MS analysis.
Experiments that use the “separate-before-digest” techniques for protein profiling generally fall into two classes. In the first class, called “global” protein profiling, a mixture of proteins is thrown at the 2DE system or a set of 2DE systems (each with a different narrow IPG) with a goal of resolving the entire proteome of the sample (or as much as was able to load on the 2DE system) and detecting, excising, digesting, and identifying the interesting spots with MALDI-MS. While this approach is simple and powerful and (when running multiple narrow range 2D gels on the same sample) has been reported to resolve more than 10000 proteins from a higher eukaryotic cell lysate, the global approach has the weakness that no little or no functional information is revealed by the separation itself and high abundance proteins are known to mask low abundance proteins. Additionally, the need for extremely high resolution in global methods requires the use of 2DE separation which has some known difficulties in its own right in the areas of: handling low abundance proteins (because overloading causes loss of resolution and high abundance proteins can mask low abundance spots); extreme pI proteins (less than 3 and more than 9); cysteine rich proteins (which often smear under isoelectric focusing); and a general difficulty in reproducing 2DE patterns from lab to lab.
An alternative to the “global” approach is a “fractionated” protein profiling approach, which generally involves using affinity columns, bead precipitation and/or selective labeling to fractionate the initial protein sample into one or more sub-proteomes. The sub-proteomes are then separated, digested and identified as in the global approach. Advantages of the “fractionated” approach include the fact that a protein is a member of the selected fraction can be very revealing as to its function or its state.
An example of the fractionated protein profiling approach is Post-Translational Modification (PTM). Several groups have isolated proteins phosphorylated on either tyrosine or serine/threonine residues. By construction, all bands in these experiments have undergone some post translational modification, and in sample difference experiments a band appearing or disappearing directly indicates phosphorylation modulated signaling in those samples.
Another example of fractionated protein profiling is Activity-Based Protein Profiling (ABPP). By employing probes that covalently link to an enzyme active site fractions of “active” enzyme families have been isolated for separation.
Yet another example of fractionated protein profiling is Protein—Protein interaction. By binding “bait” proteins to beads and isolating the fraction of proteins that bind to the bait many groups have studied protein—protein interactions.
A final example is Sub-Proteome Elution. Using an elution column of heparin-sepharose whole cell proteomes of CHO and RCC cells can be fractionated into 3 sub-proteomes.
In the fractionated approach, the problem of identifying the proteins in a sub-proteome is reduced by reducing the number of proteins that are separated from thousands of proteins to less than about a hundred proteins (depending on how stringent the fractionation is). This both allows increased loading of the proteins that remain in the fraction (relative to the amount of those proteins that would be loadable if part of an unfractionated mixture) and removes a large quantity of possibly masking proteins. In many cases the separation problem is reduced enough to be tractable with one-dimensional electrophoresis (1DE) instead of 2D, which allows one to take advantage of the superiority of 1DE, including greater loading capacity, better ability to handle large protein complexes (above 100 kDa), better ability to handle extreme pI and hydrophobic proteins, better quantitation, and generally more reliability and consistency from run-to-run. In some cases enough information is gleaned from the labeling and fractionation to make it unnecessary to go to MS.
A “fractionated” approach to protein expression analysis then has some real advantages in terms of information content, reliability, sensitivity and spectrum of proteins that can be handled. However, the fractionated approaches to protein expression analysis require a manual affinity purification step before a separation procedure, which is not very well defined or automatable. Current fractionated approaches therefore are slow, with, at best, procedure of a least on day in duration to go from gel to MALDI-MS.